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Grp94 inhibitors

a technology of inhibitors and grp94, which is applied in the field of grp94 inhibitors, can solve the problems of non-selective inhibition, no isoform selective inhibitors have yet been discovered, and multiple detriments,

Active Publication Date: 2013-05-02
UNIVERSITY OF KANSAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides novel compounds that selectively inhibit Grp94, a heat shock protein involved in cancer, inflammation, and neurodegenerative disorders. These compounds are selective for Grp94 over other Hsp90 isoforms and can be used as anti-cancer, anti-inflammatory, and neuroprotective agents. The compounds have a unique structure that allows for the inhibition of Grp94 without affecting other proteins. The invention also provides pharmaceutical compositions and methods for treating or preventing Grp94-related disorders in patients.

Problems solved by technology

Although Hsp90 inhibition has garnered tremendous attention in drug development for multiple disease states and numerous companies are in late-stage clinical development, no isoform selective inhibitor has yet been discovered.
Multiple detriments are associated with current Hsp90 inhibitors, which in many cases result from the non-selective inhibition of all four Hsp90 isoforms.
Although Hsp90 represents a promising therapeutic target for the treatment of cancer and other diseases, unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed.

Method used

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Examples

Experimental program
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Effect test

example 1

Synthesis of GRP94 Inhibitors

[0226]General Method for the Synthesis of Compounds 1-5. Aldehyde 6 (1 equiv.) was dissolved in wet MeOH at 25° C. The required aniline / amine (1 equiv.) was added dropwise via a syringe to the reaction flask followed by addition of ammounim bicarbonate (1 equiv.). Glyoxal (1 equiv.) was then added dropwise via a syringe and the reaction was allowed to stir at 25° C. for 8 h. Upon complete conversion of the aldehyde, as observed by thin-layer chromatography, tetrabutylammonium fluoride was added dropwise via syringe and the reaction was allowed to stir at 25° C. for 30 min, at which time, the reaction was quenched with sat. aq. NH4Cl and extracted with EtOAc. The organic layers were combined, dried over Na2SO4, and concentrated in vacuo. All compounds were purified via flash chromatography utilizing 95:5 (CH2Cl2:MeOH) as the eluent. Yields and characterization for all compounds are provided below.

Compound 2, Methyl 2-(2-(1-benzyl-1H-imidazol-2-yl)ethyl)-3...

example 2

Cell Culture

[0243]HEK293 and C2C12 cells were maintained in DMEM supplemented with non-essential amino acids, L-glutamine (2 mM), streptomycin (500 μg / mL), penicillin (100 units / mL), and 10% FBS. Cells were grown to confluence in a humidified atmosphere (37° C., 5% CO2). Stable GRP94-siRNA knockdown cell lines were generated as folllows: the shRNA sequence 5′-GGCUCAAGGACAGAUGAUGtt-3′ was cloned into the A pSilencer 2.0-U6 vector (Ambion) and positive clones confirmed by sequencing. The pSilencer 2.0-U6-GRP94 siRNA vector and a control, non-targeting pSilencer 2.0-U6 siRNA vector (scrambled, control) were transfected into HEK293 cells using Lipofectamine 2000 and the manufacturers protocol. Cell cultures were selected thirty six hours post-transfection by addition of 1 microgram / ml puromycin to the media. Puromycin resistant clones (both GRP94 siRNA and non-targeting siRNA) were subsequently expanded and screened for knockdown efficiency by immunoblotting, using GRP94 antibody DU120....

example 3

Toll Trafficking Assay Protocol

[0244]Prior studies by Nicchitta have shown that the Toll receptors are solely dependent upon GRP94 for their trafficking to the cell membrane, as illustrated in FIG. 4. Thus, they have used surface expression of Toll to monitor GRP94-mediated trafficking, which in the presence of siRNA targeting GRP94, little to no Toll is presented at the cell surface. HEK293 cells stably expressing a GRP94 siRNA or scrambled siRNA are transfected with a plasmid encoding the expression of TOLL, the drosophila homologue of the human unterleukin 1 receptor. After transfection, cells are treated with Hsp90 inhibitors for 24 hours. After drug tteatment the surface expression of Toll is monitored by either fluorescence microscopy or flow cytometry. The trafficking of Toll to the cell surface is dependent on GRP94. Cells deficient in GRP94 (i.e. GRP94 siRNA) or cells that have been treated with GRP94 inhibitor will show reduced expression of Toll. Western blots are then us...

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PUM

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Abstract

The present disclosure provides a series of compounds which exhibit isoform selective inhibition of GRP94, a homologue of Hsp90 that is localized to the endoplasmic recticulum. Through GRP94 inhibition, these compounds are likely to manifest anti-cancer, anti-inflammatory, anti-metastasis, and immunosuppressive activities, as well as utility in the treatment of neurodegenerative diseases, and diabetes.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims the benefit of priority to U.S. Provisional Application Ser. No. 61 / 473,343, filed Apr. 8, 2011, which is hereby incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with Government support under National Institutes of Health (NIH) Grant Nos. AG18001, GM077480, DK053058 and CA109265, awarded by the National Cancer Institute. The Government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present disclosure provides a series of compounds which exhibit isoform selective inhibition of Glucose-related protein 94 (Grp94), a homologue of the cytoplasmic Heat shock protein 90 (Hsp90) that is localized to the endoplasmic recticulum. Through Grp94 inhibition, these compounds are likely to manifest anti-cancer, anti-inflammatory, anti-metastasis, and immunosuppressive activities, in addition to ex...

Claims

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Application Information

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IPC IPC(8): C07D233/64C07D265/36C07D207/27C07C69/84C07D401/06C07D403/06
CPCC07D233/26C07D401/06C07D403/06A61K31/415C07D207/27C07D233/64C07D265/36C07C69/84
Inventor BLAGG, BRIAN S.J.DUERFELDT, ADAM S.
Owner UNIVERSITY OF KANSAS
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